The long-term goal of this research program is to understand the regulation of the NO signaling pathway and its role in the mechanisms of anesthesia and analgesia. PSD-95/SAP9O is one of a family of proteins recently shown to physically link other proteins (e.g. NMDA receptor and neuronal NO synthase) together into macromolecular structures via PDZ domain interactions at the synapse. This proposal is focused on the interaction of PSD-95/SAP90 with the excitatory neurotransmitter-NO signaling pathway in spinal analgesic and anesthetic mechanisms. We have made the novel discovery that the PSD-95/SAP9O protein interacts with both NMDA receptors and neuronal NOS in the spinal cord, and that suppression of the expression of PSD-95/SAP90 protein both significantly attenuated spinal hyperalgesia triggered by NMDA receptor activation and reduced the requirement for inhalational anesthetics. The current proposal seeks to further define the role of PSD-95/SAP90 in spinal analgesia and anesthesia, to understand the molecular mechanisms of spinal analgesia resulting from suppressing PSD-95/SAP90 expression, and to further determine the molecular mechanisms of interaction of inhalational anesthetics with the excitatory neurotransmitter-NO signaling pathway. This proposal will determine whether suppression of the expression of PSD-95/SAP90 affects the development of hyperalgesia in formalin and neuropathic models of pain, and the responses of spinal dorsal horn neurons to noxious stimulation. Ultra structural localization of PSD- 95/SAP90 and its synaptic relationship with glutamate, neuronal NOS and GABA in the spinal cord will be determined in order to define synaptic mechanisms of PSD-95/SAP90's action. The effect of suppressing PSD- 95/SAP90's expression on the NO-cGMP signaling pathway via NMDA receptor activation in the spinal cord will be investigated to define whether the NO signaling pathway is one of the downstream pathways by which PSD-95/SAP90 couples NMDA receptor activity to spinal cord pad mechanisms. To further define the role of PSD-95/SAP90 in the anesthetic state, studies will be performed to observe dose-dependent changes in requirement for inhalational anesthetics and in locomotor activity in rats with a deficiency of PSD-95/SAP90 expression. The molecular effect of inhalational anesthetics on the PDZ domain interaction between PSD- 95/SAP90 and NMDA receptors of neuronal NOS will be investigated to elucidate the mechanism of anesthetic inhibition of the NO signaling pathway.